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Sirna mediated gene silencing in transgenic animals

Inactive Publication Date: 2006-09-28
ARTEMIS PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011] The living organisms in embodiments (1) to (3) above are preferably vertebrates. In embodiments (2) and (3) the living organisms are more preferably non-human vertebrates. The ti

Problems solved by technology

Although RNAi in mice has been in principle demonstrated, the current technology does not allow performing systematic gene function analysis in vivo.
Although these results demonstrate that the mechanism of shRNA mediated gene silencing is functional in mice, they do not clarify whether constitutive RNAi can be achieved in transgenic animals.
These experiments included random integration of shRNA transgenes and screening for clones with appropriate siRNA expression, which is not applicable for testing of a large number of different shRNA transgenes in mice.
However, these experiments again included random integration of shRNA transgenes resulting in variable levels and patterns of shRNA expression.
Thus, testing of ES cell clones or mouse lines with appropriate shRNA expression had been required, which is a laborious and time-consuming undertaking.
It has been, however, questionable whether a single copy of a siRNA expression vector integrated into the genome would result in sufficiently high levels of siRNA required for RNAi-mediated gene inhibition in multiple organs of the living organism.
However, it has been unpredictable for a person skilled in the art which genomic region within the living organism promotes an appropriate expression pattern of the Pol III promoter driven shRNA constructs required for RNAi-mediated gene inhibition in multiple organs of the living organism.
It is, however, not obvious whether a stably integrated, single copy configuration of these systems can be created that allows inducible RNAi in multiple organs without background activity.

Method used

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  • Sirna mediated gene silencing in transgenic animals
  • Sirna mediated gene silencing in transgenic animals
  • Sirna mediated gene silencing in transgenic animals

Examples

Experimental program
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Effect test

example 1

[0061] The firefly luciferase gene along with a splice acceptor sequence (Friedrich and Soriano, Genes Dev. 9:1513-23 (1991)) was inserted into the first allele of the rosa26 locus by homologous recombination in ES cells. The shRNA expression cassettes under the control of the H1 or the U6 promoter and a Renilla luciferase gene were inserted into the second allele of the rosa26 locus by homologous recombination. The Renilla luciferase gene was used as a reference to normalize the values of firefly luciferase activity.

[0062] Deleting the loxP-flanked shRNA expression cassette by transient transfection of a cre expression plasmid generated the negative control. FIG. 7 shows the expression of the firefly luciferase in the absence and the presence of the shRNA expression cassette. Expression of the shRNA under the control of the H1 promoter resulted in a ˜75% reduction of firefly luciferase activity, whereas the repression mediated by the U6-shRNA construct was ˜60%.

example 2

[0063] The shRNA expression cassette under the control of the U6 promoter containing tet-operator sequences, and a Renilla luciferase gene were inserted into the first allele of the rosa26 locus (FIGS. 8 and 12; SEQ ID NOs:9 and 10). The firefly luciferase gene under the control of the endogenous rosa26 promoter and a tet repressor expression cassette (Gossen and Bujard, PNAS. 89: 5547-5551 (1992)) was inserted into the second allele of rosa26 by homologous recombination in ES cells. (FIG. 8; SEQ ID NO:14). To determine the extent of shRNA-mediated gene silencing, the cells were treated for 2 days in the presence or in the absence of 1 μg / ml Doxycycline in the cell culture medium. FIG. 10 shows the firefly luciferase activity measured in the cell extracts. Doxycycline inducible expression of the shRNA under the control of the U6tetO1 promoter (SEQ ID NO:9) resulted in a ˜30% reduction of firefly luciferase activity, whereas repression mediated by the U6tetO2-construct (SEQ ID NO 10)...

example 3

[0064] NIH3T3 cells were transiently transfected with constructs expressing the luciferase and tet repressor genes (SEQ ID NOs:15 and 16) together with the shRNA constructs under the control of the H1 promoter containing tet-operator sequences (sequence ID NOs:11, 12 and 13; FIG. 9). FIG. 11 shows the expression of firefly luciferase in the absence and in the presence of 1 μg / ml doxycycline. The highest degree of doxycycline inducible shRNA expression was achieved with the H1tetO2 / 3′-promoter, resulting in a ˜80% reduction of firefly luciferase activity.

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Abstract

The present invention relates to a process that enables constitutive and inducible gene knock down in living organisms using a short hairpin RNA expression vector integrated into the genome of the organism.

Description

[0001] The present invention relates to a process that enables constitutive and inducible gene knock down in living organisms using a short hairpin RNA expression vector integrated into the genome of the organism. BACKGROUND OF THE INVENTION [0002] RNA interference (RNAi) has been discovered some years ago as a tool for inhibition of gene expression (Fire, A. et al., Nature 391, 806-811 (1998)). It based on the introduction of double stranded RNA (dsRNA) molecules into cells, whereby one strand is complementary to the coding region of a target gene. Through pairing of the specific mRNA with the introduced RNA molecule, the mRNA is degraded by a cellular mechanism. Since long dsRNA provokes an interferon response in mammalian cells, the technology was initially restricted to organisms or cells showing no interferon response (Bass, B. L., Nature 411, 428-429 (2001)). The finding that short (<30 bp) interfering RNAs (siRNA) circumvent the interferon response extended the application...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N15/11C12N15/63C12N15/85
CPCA01K2227/40A01K2267/03A61K48/00C12N15/111C12N15/63C12N15/8509C12N2310/111C12N2310/14C12N2330/30C12N2800/30A01K67/0275A01K2217/05A01K2217/058A01K2217/20A01K2227/105
Inventor SEIBLER, JOSTSCHWENK, FRIEDERKÜHN, RALFKÜTER-LUKS, BIRGIT
Owner ARTEMIS PHARMA